The IL-6 receptor super-antagonist Sant7 enhances antiproliferative and apoptotic effects induced by dexamethasone and zoledronic acid on multiple myeloma cells

Interleukin-6 (IL-6) is the major growth and survival factor for multiple myeloma (MM), and has been shown to protect MM cells from apoptosis induced by a variety of agents. IL-6 receptor antagonists, which prevent the assembly of functional IL-6 receptor complexes, inhibit cell proliferation and induce apoptosis in MM cells. We have investigated whether the IL-6 receptor super-antagonist Sant7 might enhance the antiproliferative and apoptotic effects induced by the combination of dexamethasone (Dex) and zoledronic acid (Zln) on human MM cell lines and primary cells from MM patients. Here we show that each of these compounds individually induced detectable antiproliferative effects on MM cells. Sant7 significantly enhanced growth inhibition and apoptosis induced by Dex and Zln on both MM cell lines and primary MM cells. These results indicate that overcoming IL-6 mediated cell resistance by Sant7 potentiates the effect of glucocorticoides and bisphosphonates on MM cell growth and survival, providing a rationale for therapies including IL-6 antagonists in MM.     

Combination of interferon and dexamethasone in refractory multiple myeloma

The present pilot study was designed to analyse the efficacy and toxicity of the association of interferon (IFN) and dexamethasone (Dx) in 32 resistant and relapsed myeloma patients. Among the evaluable cases, 15 (68 per cent) responded to treatment (32 per cent achieved an objective response--OR--and 36 per cent a partial response--PR--), the 'remission' status lasting for more than one year in six of them. Moreover, four out of the seven OR patients showed a reduction in B.M. plasma cells to less than 5 per cent. Five out of 11 patients that were previously refractory to VBAD, that includes high dose dexamethasone (Dx), responded to IFN-Dx. The protocol was generally well tolerated with only four patients discontinuing therapy due to adverse effects. The present results show that the combination IFN + Dx is a promising therapeutic approach for patients with refractory myeloma.     

沙利度胺联合地塞米松治疗复发和(或)难治及平台期多发性骨髓瘤疗效分析

目的观察沙利度胺联合地塞米松（TD方案）治疗多发性骨髓瘤（MM）的疗效。方法62例MM患者,其中复发和（或）难治组25例,平台期组37例。复发和（或）难治组治疗方案为：TD方案3个疗程后无效或进展者更换方案;有效者,继续使用TD方案,3个疗程后停用地塞米松,单独使用沙利度胺直到复发。平台期组的患者仅使用3个疗程的TD方案,再单独使用沙利度胺维持治疗。结果25例复发和（或）难治的患者,前3个疗程TD方案的25例中20例总有效[非常好的部分缓解（VGPR）＋部分缓解（PR）＋进步（MR）]率为80％,但无完全缓解（CR）或接近完全缓解（nCR）。有效者,经后3个疗程TD治疗后,1例获得nCR,而2例PR患者回到MR,无患者发展到NR或进展;对13例VGPR＋PR＋nCR患者,单独使用沙利度胺4～12个月（中位时间6．8个月）后复发。37例平台期的患者经上述方案治疗8～26个月（中位时间17．5个月）后复发。明显优于难治和（或）复发组的治疗效果（P〈0．001）。结论沙利度胺联合地塞米松是难治和（或）复发MM有效治疗方案,也可作为平台期患者的维持治疗。     

Combination therapy of Thalidomide and Peginterferon in patients with progressive multiple myeloma.

The rationale of this study was to investigate the safety andtherapeutic effect of a combination therapy of Thalidomide (Thal)and IFN-. Both agents are known to exert immunomodulatory andanti-angiogenic effects and are used for therapy in multiplemyeloma (MM) [1]. In a phase I/II study, Thal was started ata dose     

Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma

Multiple myeloma (MM) is a hematologic malignancy of human plasma cells, and myeloma cells can be classified into several subpopulations according to phenotypic differences, such as CD38 MPC-1- CD49e- immature, CD38 MPC-1+ CD49e- intermediate and CD38 MPC-1+ CD49e+ mature myeloma cells. The expression of the CD45 molecule on myeloma cells is quite variable, and the physiological consequence of CD45 on myeloma cells is still unknown. Recently, we have found that a few MPC-1- immature myeloma cells express CD45 antigens while most myeloma cells do not express the CD45. MPC-1- CD45+ CD49e- but not MPC-1- CD45- CD49e- immature cells contain proliferating cells in response to interleukin-6 (IL-6). IL-6 can also induce expression of CD45 on the MPC-1- CD45- subpopulation of immature myeloma cells. In addition, myeloma cell lines responding to IL-6 express CD45, whereas cell lines proliferating independent of IL-6 do not express CD45. In the U266 cell line, IL-6 leads to the induction of CD45 expression and cell proliferation, indicating that IL-6-induced effects are closely linked to CD45 expression. Thus, there is a heterogeneity in human myeloma cells, and among these subpopulations immature myeloma cells expressing the CD45 molecules appear to proliferate in response to IL-6. In this review we propose the involvement of CD45 in MM pathogenesis, and the possible implications of CD45 as both a phenotypic marker and a functional molecule is discussed.     

The relationship between soluble receptor of interleukin-6 with angiogenic cytokines and proliferation markers in multiple myeloma

Soluble interleukin-6 receptor (sIL-6R) is part of IL-6 receptor that may stimulate cells that do not express the whole molecule. It may enhance myeloma cell proliferation and furthermore angiogenesis. The aim of the study was to evaluate the clinical significance and the relationship between serum levels of sIL-6R, with various stimulators of angiogenesis, such as hepatocyte growth factor (HGF) and interleukin-18 (IL-18) and with markers of proliferation, such as beta-2 microglobulin (B2M) levels and plasma cell Ki-67 proliferation index in the bone marrow, in patients with multiple myeloma (MM). We studied 45 newly diagnosed MM patients. Serum levels of sIL-6R, HGF, IL-18, and B2M and Ki-67 proliferation index (Ki-67 PI) in bone marrow’s plasma cells were determined. The mean concentrations of sIL-6R, HGF, IL-18, and B2M and the value of Ki-67 were significantly higher in the patients compared to controls and with increasing disease stage. sIL-6R was strongly positively correlated with HGF, IL-18, B2M, and Ki-67 PI. There is a positive correlation between plasma cell growth, as determined by Ki-67 PI, and different angiogenic cytokines, such as HGF and IL-18, with sIL-6R. This relationship suggests the significant role of these cytokines in the proliferation and disease activity in MM patients.     

In vitro growth of human multiple myeloma: implications for biology and therapy.

In vitro data allow presentation of a plausible scenario for the in vivo growth, progression, and dissemination of human multiple myeloma (MM) that involves the interactions between the monoclonal B-cell clone and the bone marrow (BM) microenvironment. A large series of adhesion and extracellular matrix molecules allow trapping of circulating plasma cell precursors within the BM, and a battery of locally released cytokines promote their growth and final differentiation. Malignant B cells establish close contacts with BM stromal cells and release a host of cytokines that recruit and activate BM stromal cells and also T lymphocytes to produce other cytokines. All these cytokines might conceivably act in concert in a self-perpetuating mechanism of mutual help between malignant plasma cells and BM stromal cells to favor the progressive expansion of the malignant clone through a sort of an "avalanche effect." Also, most cytokines produced by malignant B cells, stromal cells, and activated T lymphocytes, including IL-1 beta, TNF-beta, M-CSF, IL-3, and IL-6, have osteoclast-activating properties, thus explaining why the expansion of the B-cell clone is matched by the activation and numeric increase of osteoclasts.     

Recombinant interleukin-12 and interleukin-18 antitumor therapy in a guinea-pig hepatoma cell implant model

Interleukin (IL)-12 and IL-18 are secreted by myeloid cells activated with adjuvants such as Bacillus Calmette-Guérin (BCG) cell wall. They induce T-helper 1 polarization in the host immune system and upregulate production of lymphocyte interferon-γ, which leads to the induction of an antitumor gene program. It has been reported that humans have an immune system that more closely resembles that of the guinea pig in adjuvant-response features rather than the mouse system, which prevents the mouse results being extrapolated to human immunotherapy. Here we have constructed a tumor-implant system in guinea pigs to evaluate the antitumor potential of guinea pig IL-12 (gpIL-12) and guinea pig IL-18 (gpIL-18). Purified recombinant gpIL-12 and gpIL-18 were prepared and applied intraperitoneally to tumor-bearing (line 10 hepatoma) guinea pigs as the basis of the adjuvant immunotherapy. Intraperitoneal administration of gpIL-12 and gpIL-18 led to retardation of primary tumor growth and suppression of lymph-node metastasis in tumor-bearing guinea pigs. The permissible range of IL-12 appeared wider in guinea pigs than in mice. Even at an IL-12 dose higher than that in mice, there was no evidence of side-effects until day 26, when the guinea pigs were killed. gpIL-18 augmented the antitumor effect of gpIL-12 but exerted less ability to suppress lymph-node metastasis. The effects of gpIL-12 and gpIL-18 on the tumors implanted in guinea pigs will encourage us to use IL-12- and IL-18-inducible adjuvants for immunotherapy in human patients with solid cancer. ( Cancer Sci 2007; 98: 1936–1942)     

Combination effects of etoposide with other antitumor drugs in vitro and in vivo

Combination effects of etoposide (ET) with each of 10 antitumor drugs were examined with P 388 leukemia cells in vitro and in vivo. Median effect analysis was applied for the evaluation of in vitro effect by the growth inhibition, and the in vivo effect by comparison of the increase of life span (ILS) in a combined group with the sum of ILS's in 2 single agent groups. Among 10 drugs combined with ET, cyclophosphamide (melphalan was used for in vitro study), cisplatin and 6-mercaptopurine exhibited a strong synergism both in vitro and in vivo. The combination of ET with mitomycin C, vincristine, vindesine or cytarabine produced additive or slightly synergistic effect in the both systems. However, methotrexate + ET combination showed an antagonistic effect. Although the combination of ET with doxorubicin or fluorouracil showed a slight synergism in vitro, it was antagonistic in vivo. Thus, the in vitro and in vivo combined effects were consistent in 8 of 10 drugs. The employed methods in the present studies could distinguish high efficacy of ET + cyclophosphamide and ET + cisplatin, which are clinically approved as effective combinations against lung cancer. The methods seem to be useful to assess the drug efficacy in experimental combination.     

A novel isocoumarin derivative induces mitotic phase arrest and apoptosis of human multiple myeloma cells

Purpose The isocoumarin NM-3 reverses resistance of human multiple myeloma (MM) cells to dexamethasone and is in clinical trials. In the present work, the NM-3 analog, 185322, has been studied for activity against MM cells. Methods Human U266, RPMI8226 and primary MM cells were analyzed for the effects of 185322 on cell cycle distribution, tubulin polymerization and induction of apoptosis. Results We show that, in contrast to NM-3, treatment with 185322 is associated with a marked arrest of MM cells in M phase. The results also demonstrate that treatment with 185322 is associated with a rapid decrease in tubulin assembly and an increase in Bcl-2 phosphorylation, consistent with disruption of mitosis. Our results further demonstrate that mitotic failure induced by 185322 results in activation of an apoptotic response in MM cell lines and primary MM cells. By contrast, 185322 had little if any effect on growth and survival of human carcinoma cells. Conclusion These findings identify a novel inhibitor of microtubule assembly that induces mitotic arrest and apoptosis of MM cells.     

Ketoconazole potentiates the antitumor effects of nocodazole: In vivo therapy for human tumor xenografts in nude mice

Our previous studies demonstrated that the oral antifungal agent ketoconazole (KT) induces apoptosis and G0/G1 phase cell cycle arrest in human cancer cell lines. In this study, we first demonstrated that KT (1 microM) potentiated the apoptotic effects of nocodazole (ND, 1 nM) in COLO 205 cancer cells. We further demonstrated the therapeutic efficacy of a combined treatment of KT (50 mg/kg/three times per week) and ND (5 mg/kg/three times per week) in vivo by treating athymic mice bearing COLO 205 tumor xenografts. The antitumor effects of ND were significantly potentiated by KT in mice after 6 wk of treatment. No gross signs of toxicity were observed in mice receiving these treatment regimens. The apoptotic cells were detected in a microscopic view of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and by observation of DNA fragmentation in KT + ND-treated tumor tissues. The levels of cell cycle regulatory proteins were determined by Western blot analysis. Treatment with KT inhibits tumor growth through elevation of p53, p21/CIP1, and p27/KIP1 as well as inhibition of cyclin D3 and cyclin-dependent kinase 4 protein expression. Immunohistochemical staining analysis showed that p53, p21/CIP1, and p27/KIP1 immunoreactivity were induced in the tumor tissues. To clarify the roles of the p21/CIP1 and p27/KIP1 protein expression involved in G(0)/G(1) arrest and/or apoptosis induced by a combined treatment with KT and ND, antisense oligodeoxynucleotides (ODNs) specific to p21/CIP1 and p27/KIP1 were used. Our results demonstrated that apoptotic phenomena, including BAX induction and cytochrome C released from mitochondria induced by KT + ND, were significantly attenuated by pretreatment the cells with the p27/KIP1-specific antisense ODNs. These results indicate that p27/KIP1 protein does indeed play a critical role in the KT + ND-induced apoptosis. Our study revealed the molecular mechanism of KT + ND in regression of the tumor growth. The apoptotic effects of KT in a great variety of cancer cells make it a very attractive agent for cancer chemotherapy.     

In vivo and in vitro enhanced antitumor effects by pentoxifylline in human cancer cells treated with thiotepa

Methylxanthines enhance lethality of alkylating agents in human cancer cells, a phenomenon attributed to the prevention of DNA repair. Pentoxifylline is a nontoxic methylxanthine, used clinically for claudication. Using human cancer cells in culture or in a mouse xenograft model, we studied combination treatments with alkylating agents and pentoxifylline or other methylxanthines. With human bladder cancer cells in culture, cytotoxicity of thiotepa was increased up to 10-fold (P less than 0.01) by posttreatment with pentoxifylline, with a major clinical metabolite of pentoxifylline, or with caffeine; the pentoxifylline concentrations required (0.4-1.0 mM) are clinically achievable in the bladder after nontoxic p.o. doses. With human bladder or breast cancer xenografts in a modified subrenal capsule assay, enhancement of thiotepa was also observed by in vivo posttreatment with pentoxifylline. In contrast, these combinations produced no increased toxicity to normal tissues in these animals, measured by weight, lethality, or histological changes of the normal bladder urothelium. These results provide evidence for a novel approach to improve the therapeutic index of thiotepa and other alkylators, used for topical therapy of bladder cancer and, possibly, systemic therapy of other malignancies.     

In vivo induction of insulin-like growth factor-I receptor and CD44v6 confers homing and adhesion to murine multiple myeloma cells

One of the main characteristics of multiple myeloma (MM) cells is their specific homing and growth in the bone marrow (BM). Differences between stroma-dependent and -independent MM cell lines may reveal key molecules that play important roles in their homing to the BM. We addressed this topic with a murine MM model, including the in vivo 5T33MM (5T33MMvv) stroma-dependent cell line and its in vitro stroma-independent variant (5T33MMvt). Fluorescence-activated cell-sorting analysis showed expression of insulin-like growth factor (IGF)-I receptor and CD44v6 on all 5T33MMvv cells but not on 5T33MMvt cells. Checkerboard analysis and adhesion assays revealed IGF-I-dependent chemotaxis toward BM-conditioned medium and involvement of CD44v6 in the adhesion to BM stroma of only 5T33MMvv cells. However, when 5T33MMvt cells were injected in vivo (5T33MMvt-vv), after 18 h the MM cells harvested from BM were IGF-I receptor and CD44v6 positive. This up-regulation was confirmed in 5T33MMvt-vv cells isolated from terminally diseased animals. These ST33MMvt-vv cells exhibited IGF-I-dependent chemotaxis and CD44v6-dependent adhesion to BM stroma. In vitro culture of the 5T33MMvt-vv cells could completely down-regulate IGF-I receptor and CD44v6. In fact, we could show that direct contact of 5T33MMvt cells with BM endothelial cells is a prerequisite for IGF-I receptor and CD44v6 up-regulation. These data indicate that the BM microenvironment is capable of up-regulating molecules such as IGF-I receptor and CD44v6, which facilitate homing of MM cells to the BM and support their adhesion to BM stroma.     

沙利度胺对骨髓瘤细胞IL-6及其传导途径的影响

目的　探讨沙利度胺治疗难治复发性多发性骨髓瘤 (MM)的有效机制。方法　用双抗夹心法 (ELISA)动态检测MM患者血清白细胞介素 6 (IL 6 )水平 ,流式细胞仪检测瘤细胞表面IL 6受体 (IL 6R)的表达 ,RT PCR半定量法检测IL 6Rβ亚单位mRNA的表达。 结果　口服 2 0 0mg/d沙利度胺前 ,MM患者血清IL 6水平为 5 6 4.8± 319.4ng/L ,瘤细胞表面IL 6R阳性率为 33.6 % ;口服 2 0 0mg/d沙利度胺后第 14天 ,IL 6水平为 5 6 0 .3± 414.8ng/L ,瘤细胞表面IL 6R阳性率为 31.8% ,分别与口服沙利度胺前比较 ,差异无显著性 (P >均 0 .0 5 )。口服 40 0mg/d沙利度胺第 14,2 8,42 ,5 6 ,84天 ,IL 6水平分别为 5 16 .7± 131.9、42 6 .7± 180 .4、387.9± 187.4、35 0 .1± 85 .5和 2 12 .3± 92 .5ng/L ,瘤细胞表面IL 6R阳性率分别为 2 8.5 %、2 4.3%、2 1.3%、12 .6 %和 10 .1% ,均分别低于口服 2 0 0mg/d沙利度胺前IL 6水平或瘤细胞表面IL 6R阳性率 (P <0 .0 5或P <0 .0 1) ;口服 2 0 0mg/d沙利度胺前及口服第 14天的IL Rβ亚单位mRNA比值分别为 7.8和 6 .9,二者差异无显著性 (P >0 .0 5 ) ;口服沙利度胺 40 0mg/d第 14,2 8天IL Rβ亚单位mRNA的比值分别为 5 .3和 2 .7,与口服 2 0 0mg/d沙利度胺前的比较 ,差异有显著性 (P     

Interleukin-6 and JAK2/STAT3 signaling mediate the reversion of dexamethasone resistance after dexamethasone withdrawal in 7TD1 multiple myeloma cells

We previously reported the establishment and characteristics of a DXM-resistant cell line (7TD1-DXM) generated from the IL6-dependent mouse B cell hybridoma, 7TD1 cell line. After withdrawing DXM from 7TD1-DXM cells over 90 days, DXM significantly inhibited the cell growth and induced apoptosis in the cells (7TD1-WD) compared with 7TD1-DXM cells. Additionally, IL-6 reversed while IL-6 antibody and AG490 enhanced the effects of growth inhibition and apoptosis induced by DXM in 7TD1-WD cells. Our study demonstrates that 7TD1-DXM cells become resensitized to DXM after DXM withdrawal, and IL-6 and JAK2/STAT3 pathways may regulate the phenomenon.